This disclosure relates to an isolated genetic regulatory sequence specific to neural progenitor cells and methods to use said regulatory sequence to mark, isolate, and manipulate neural progenitors.
Stem cell research in the central nervous system holds great promise for the development of novel therapies for brain damage and other human diseases. Researchers are keenly interested in adult stem cells because they do not pose the ethical questions raised by embryonic stem cells. One of the ultimate goals in adult stem cell research is to better understand the biology of these cells so that scientists may develop strategies to directly manipulate them within the damaged brain.
In the dentate gyrus (DG) of the hippocampus, new neurons continue to be born from resident stem/progenitor cells at the subgranular zone (SGZ) throughout our lives (Altman and Das, 1965; Cameron et al., 1993; Eriksson et al., 1998; Kornack and Rakic, 1999). Adult-generated granular neurons are indistinguishable from neighboring granular neurons in terms of their morphology, synaptic connections, and electrophysiological properties (Cameron et al., 1993; Hastings and Gould, 1999; Markakis and Gage, 1999; van Praag et al., 2002). Thus, it is likely that these adult-generated cells functionally participate in learning and memory.
At least three partially overlapping cell populations have been identified at adult SGZ based on their distinct morphologies and expression of molecular markers (Ehninger and Kempermann, 2008; Zhao et al., 2008). Type-1 progenitors (or neural stem-like cells, Type B cells (Seri et al., 2001)), which rarely divide, can be identified by their expression of glial fibrillary acidic protein (GFAP) and nestin (Fukuda et al., 2003) and by their radial processes with highly elaborated arbors branching into the molecular layers (Mignone et al., 2004). A nuclear transcription protein Sox2 has also been recognized as a marker for mainly Type-1 cells (Steiner et al., 2006). There are at least two types of transient amplifying progenitors. Type-2 cells (or Type D cells (Seri et al., 2001)), are most highly proliferative cells at SGZ, have short horizontal processes; they express transcription factor Tbr2 (T-box brain gene 2) (Hodge et al., 2008) and low-level neuronal-lineage markers such as doublecortin (DCX). Type-3 progenitors represent slow-proliferating cells committed to the neural fate, and during their transit to exit cell cycle to become postmitotic neurons. These cells are highly variable in morphology and often have vertically orientated processes displaying DCX, polysialylated neural cell adhesion molecule, and TuJ1. Whereas the identity of the neural stem cells in the adult DG has been under debate (Garcia et al., 2004; Palmer et al., 1997; Seaberg and van der Kooy, 2002; Seri et al., 2001), the prevailing model has been that type-1 progenitors represent the primary precursors that give rise to transient amplifying progenitors, which subsequently differentiate into both astroglials and granule neurons (Seri et al., 2004; Seri et al., 2001).
Regulatory sequence-targeted selection has provided proof-in-principle that high-yield and high-purity of neural-lineage specific progenitors can be isolated from the adult hippocampus. However, the previously-described regulatory sequences have poor tissue-specificity. For example, the Tα-tubulin promoter also targets to neurons; GFAP promoter also targets to astroglials.
Despite the emphasis on characterizing and utilizing neural stem cells, many studies have shown that the cycling Type 2 and Type 3 progenitors, but not the Type 1 progenitors, are most responsive to various regulatory influences (Jessberger et al., 2005; Kempermann et al., 1998; Kronenberg et al., 2003) and thus are excellent candidates for manipulation and use in research, development, and medicine.
It has been recently discovered that Tctex-1 (or DYNLT1 (Pfister et al., 2005)), previously recognized as a light chain of cytoplasmic dynein (King et al., 1996), is selectively enriched in stem-like cells and cycling progenitors, but not in mature granule cells and astrocytes, in the adult dentate gyrus (DG) (Chuang et al., 2001; Dedesma et al., 2006). The SGZ-enriched Tctex-1 expression pattern was confirmed by in situ hybridization, suggesting that Tctex-1 expression is primarily regulated at the transcriptional level. Disclosed herein are the genomic sequences specifying Tctex-1 expression in the dentate progenitors of adult hippocampus and Tctex-1:GFP reporter mice in which the adult hippocampal stem-like and progenitor cells are genetically marked.
Disclosed are the genomic sequences specifying Tctex-1 expression in neural progenitors. Further disclosed are Tctex-1-GFP reporter constructs and mice in which neural progenitors and stem cells are marked.